The lizard abides: cold hardiness and winter refuges of Liolaemus pictus argentinus in Patagonia, Argentina

Abstract

In environments where the temperature periodically drops below zero, it is remarkable that some lizards can survive. Behaviorally, lizards can find microsites for overwintering where temperatures do not drop as much as the air temperature. Physiologically, they can alter their biochemical balance to tolerate freezing or avoid it by supercooling. We evaluated the cold hardiness of a population of Liolaemus pictus argentinus Müller and Hellmich, 1939 in the mountains of Esquel (Patagonia, Argentina) during autumn. Additionally, we assessed the thermal quality (in degree-days) of potential refuges in a mid-elevation forest (1100 m above sea level (asl)) and in the high Andean steppe (1400 m asl). We analyzed the role of urea, glucose, total proteins, and albumin as possible cryoprotectants, comparing a group of lizards gradually exposed to temperatures lower than 0 °C with a control group maintained at room temperature. However, we found no evidence to support the presence of freeze tolerance or supercooling mechanisms in this species as related to the analyzed metabolites. Instead, the low frequency of degree-days below 0 °C and temperatures never lower than −3 °C in potential refuges suggest that L.p.argentinus might avoid physiological investments (such as supercooling and freeze tolerance) by behaviorally selecting appropriate refuges to overcome cold environmental temperatures.

Full text

ARTICLE
The lizard abides: cold hardiness and winter refuges of
Liolaemus pictus argentinus in Patagonia, Argentina
N.R. Cecchetto, S.M. Medina, S. Taussig, and N.R. Ibargüengoytía
Abstract: In environments where the temperature periodically drops below zero, it is remarkable that some lizards can survive.
Behaviorally, lizards can find microsites for overwintering where temperatures do not drop as much as the air temperature.
Physiologically, they can alter their biochemical balance to tolerate freezing or avoid it by supercooling. We evaluated the cold
hardiness of a population of Liolaemus pictus argentinus Müller and Hellmich, 1939 in the mountains of Esquel (Patagonia,
Argentina) during autumn. Additionally, we assessed the thermal quality (in degree-days) of potential refuges in a mid-elevation
forest (1100 m above sea level (asl)) and in the high Andean steppe (1400 m asl). We analyzed the role of urea, glucose, total
proteins, and albumin as possible cryoprotectants, comparing a group of lizards gradually exposed to temperatures lower than
0 °C with a control group maintained at room temperature. However, we found no evidence to support the presence of freeze
tolerance or supercooling mechanisms in this species as related to the analyzed metabolites. Instead, the low frequency of
degree-days below 0 °C and temperatures never lower than −3 °C in potential refuges suggest that L. p. argentinus might avoid
physiological investments (such as supercooling and freeze tolerance) by behaviorally selecting appropriate refuges to overcome
cold environmental temperatures.
Key words: Liolaemus pictus argentinus, cold hardiness, cryoprotectants, thermal quality, refuges, Patagonia, degree-day.
Résumé : Dans des milieux où la température tombe périodiquement sous zéro, il est remarquable que certains lézards puissent
survivre. D’un point de vue comportemental, les lézards peuvent trouver des microsites pour hiverner où les températures ne
baissent pas autant que la température de l’air. D’un point de vue physiologique, ils peuvent modifier leur équilibre biochimique
pour pouvoir tolérer le gel ou l’éviter par surfusion. Nous avons évalué la résistance au froid d’une population de lézards
Liolaemus pictus argentinus Müller et Hellmich, 1939 dans les montagnes d’Esquel (Patagonie, Argentine) durant l’automne. Nous
avons en outre évalué la qualité thermique (en degrés-jours) de refuges potentiels dans une forêt d’élévation intermédiaire
(1100 m au-desseus du niveau de la mer (asl)) et dans une steppe des hautes Andes (1400 m asl). Nous avons analysé le rôle de l’urée,
du glucose, des protéines totales et de l’albumine comme cryoprotecteurs possibles, en comparant un groupe de lézards exposés
progressivement à des températures inférieuresà0°Càungroupe témoin maintenu à température de laboratoire. Nous n’avons
toutefois relevé aucune preuve associée aux métabolites analysés d’une tolérance au gel ou de mécanismes de surfusion chez
cette espèce. La faible fréquence de degrés-jours sous 0 °C et des températures qui ne tombent jamais sous −3 °C dans les refuges
potentiels donnent plutôt à penser que L. p. argentinus pourrait éviter les investissements physiologiques (comme la surfusion et
la tolérance au gel) par la sélection comportementale de refuges lui permettant de survivre à des températures ambiantes
froides. [Traduit par la Rédaction]
Mots-clés : Liolaemus pictus argentinus, résistance au froid, cryoprotecteurs, qualité thermique, Patagonie, degré-jour.
Introduction
Subzero temperatures are a dangerous challenge for most ani-
mals, and even more so for ectotherms (Cowles and Bogert 1944);
few reptile species can endure the harsh conditions of high lati-
tudes and elevations, where the landscape is covered with snow
and daylight hours are reduced in the coldest months of the year.
It is well known that the mean annual temperature decreases
with elevation and latitude (Körner 2007;Colwell et al. 2008), and
so the ability of lizards to inhabit these harsh environments
strongly depends on refuge availability rather than on the ther-
mal habitat quality of the area as a whole (Monasterio et al. 2009).
In this regard, lizards endure the temperature shifts and freezing
periods by selecting refuges that attenuate them (Weisrock and
Janzen 1999). This is the case for the Viviparous Lizard (Zootoca
vivipara (Lichtenstein, 1823)) in eastern Siberia, which needs stable
cavities made by beetles or decomposed roots to survive the cold-
est months of the year (Berman et al. 2016).
Even when environmental temperatures are above 0 °C, the
cold weather can be dangerous to a lizard in an ecological sense:
the animal might encounter temperatures that are low enough to
hinder physical performance, rendering it unable to escape pred-
ators (Christian and Tracy 1981). Cowles and Bogert (1944) defined
“critical thermal minimum” (CTmin) as the “thermal point at
which locomotor activity becomes disorganized and the animal
loses its ability to escape from conditions that will promptly lead
to its death”. When temperatures are lower than 0 °C, lizards can
Received 9 August 2018. Accepted 24 January 2019.
N.R. Cecchetto and N.R. Ibargüengoytía. Instituto de Investigaciones en Biodiversidad y Medioambiente (INIBIOMA), Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET), Quintral 1250, San Carlos de Bariloche, 8400, Argentina.
S.M. Medina. Centro de Investigación Esquel de Montaña y Estepa Patagónica (CIEMEP), Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Esquel, Chubut, 9200, Argentina.
S. Taussig. Laboratorios DiBio, Morales 645, San Carlos de Bariloche, 8400, Argentina.
Corresponding author: S.M. Medina (email: marlinmedina74@gmail.com).
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.
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respond by adopting one of two physiological mechanisms: freeze
tolerance or freeze avoidance (by supercooling). During a state of
freeze tolerance, ectotherms endure the partial conversion of
body fluids into ice for a variable amount of time. Freeze tolerance
in vertebrates has representatives from various groups, such as
anuran and urodele amphibians (Schmid 1982;Berman et al.
1984), snakes (Costanzo et al. 1988), lizards (Claussen et al. 1990),
and turtles (Costanzo et al. 1995a). There is high variability among
species and populations in the resistance to different percentages
of frozen body fluids, time frozen, and the number of freezing and
thawing episodes that individuals can tolerate (e.g., Z. vivipara;
Voituron et al. 2002;Berman et al. 2016). On the other hand,
supercooling is a mechanism by which the individual “can remain
unfrozen at temperatures below the equilibrium crystallization
temperature of its body fluids” (Costanzo et al. 1995a), thus involv-
ing less physiological stress (Costanzo et al. 2008). However,
supercooled solutions are inherently metastable, with the possi-
bility of spontaneously freezing at any temperature below its
equilibrium freezing point, representing a riskier mechanism
than freeze tolerance (Sømme 1982). In addition, since the proba-
bility of ice nucleation in supercooled solutions increases with
time and with the decrease in temperature (Salt 1966), this mech-
anism could have lethal consequences (Storey and Storey 1996).
In the highlands of the Andes in Patagonia, Argentina, the temper-
ature drops to freezing values for extended periods (from hours to
days) during the cold months of autumn and winter, where the
snowpack reaches a considerable height (>1 m). Studies regarding
the survival of lizards in Patagonia to low temperatures are related
to minimal voluntary and critical temperatures (CTmin) that liz-
ards can withstand without compromising their physical integ-
rity with physiological adjustments (e.g., Darwin’s Marked Gecko
(Homonota darwinii Boulenger, 1885); Espinoza and Tracy 1997)or
by behaviorally selecting warmer microenvironments (Phymaturus
tenebrosus Lobo and Quinteros, 2005; Ibargüengoytía 2005).
In this study, we analyzed the cold hardiness by physiological
and behavioral mechanisms of an emblematic lizard of the
Notophagus Blume forest, the Liolaemus pictus argentinus Müller and
Hellmich, 1939 (Liolaemidae). Liolaemus pictus argentinus is a vivip-
arous and insectivorous species with a wide distribution in the
Patagonian Andes of Neuquén, Rio Negro, and Chubut provinces
of Argentina (39°S–43°S and 520–1600 m above sea level (asl)). At
mid- and low elevations, L. p. argentinus can survive the colder
months using leaf litter, bushes, rocks, and nurse logs as shelters,
with coverage of the falling snow from the local canopy. Mean-
while, at higher elevations, the species can only survive the winter
buried under the loose ground under tussocks or under rocks
covered by snow. We hypothesize that L. p. argentinus must have
developed supercooling or freeze tolerance to survive during win-
ter and most of spring and autumn. We predict that after expo-
sure to cold, individuals of L. p. argentinus will show an increase in
the concentration of at least one of the selected biochemical vari-
ables (urea, glucose, total proteins) identified as cryoprotectants
in other lizard species. We discuss results in relation to the ther-
mal environment and the temperature of potential refuges in a
population located at high elevation (1100–1400 m asl, in Esquel,
Chubut province).
Materials and methods
Study areas and field methods
We captured adult males of L. p. argentinus on a mountain in
Esquel, Argentina (42°49=S, 71°15=W; 1400 m asl), during autumn
(April 2016; n= 17). Lizards were captured by hand or loop, and
field body temperature (T
b
) was measured immediately after cap-
ture using a digital thermometer (±0.1 °C; Omega 871A, type K 9
thermocouple; Omega, Stamford, Connecticut, USA) connected to
a catheter probe introduced about 1 cm inside the cloaca (Table 1).
We handled individuals by the head to avoid heat transfer. We
carried out captures with authorization from the Wild Life Service
of the Province of Chubut (permit No. 0460/16 MP; disposition
No. 11/2016). We followed the Guidelines for the Use of Live Amphibians
and Reptiles in Field and Laboratory Research of the American Society
of Ichthyologists and Herpetologists (ASIH), the Herpetologists’
League (HL), and the Society for the Study of Amphibians and
Reptiles (SSAR), as well as the regulations detailed in Argentinean
National Law No. 14346.
Thermal environment and operative temperatures
We obtained information for the thermal environment that
L. p. argentinus faces in the coldest months of the year from a
weather station near the population’s location as obtained from
the NASA database (http://power.larc.nasa.gov/). From this data-
base, we obtained values of the mean daily air temperature (2 m
above the ground) at 1000 m asl for the period between autumn
(March) and beginning of spring (September) 2016.
Subsequently, to get an idea of the challenges that the moun-
tains of Patagonia bring to this population at a microenvironmen-
tal level, we placed six lizard models connected to data loggers
(HOBO TEMP®H8, four-channel external data logger) in a variety
of potential refuges at the capture site. We set three of these
models in the mid-elevation Nothofagus forest (1100 m asl) and the
other three at the limit with the high Andean steppes (1400 m asl),
covering the extremes of the range where the selected popula-
tions roam freely on this mountain (Figs. 1A,1B). We placed mod-
els in different locations to determine the temperatures of
potential refuges in which the species might spend the winter
(i.e., buried ⬃15 cm underground; beneath rocks and logs; under
tussocks) during autumn, winter, and the beginning of spring
2016. We set data loggers to measure temperature every 30 min
and they were connected to thermistors located in the polyvinyl
chloride (PVC) pipe lizard models (1.5 cm×8cmsection), which
were then sealed at the ends with silicone (Fastix®) and painted
dull gray (18%) to mimic body size, reflectance, thermodynamics,
and shape of lizard bodies. We validated the models using a live
lizard and a model next to each other, exposing them to a se-
quence of temperatures and moving the model to mimic the differ-
ent positions that the lizard took under the various temperatures.
Next, we performed a regression between the model and the body
temperature of the lizards (regression: adjusted R
2
= 0.846, n=
2863, slope = 1.09, confidence interval = 1.05–1.14) to determine
if the model is a good indicator of the temperature a non-
Table 1. Descriptive data of snout–vent length (SVL; mm), body mass (g),
body condition index (BCI), critical minimum temperature (CTmin; °C),
and field body temperature (T
b
; °C) of Liolaemus pictus argentinus.
SVL (mm) Body mass (g) CTmin (°C) BCI Field T
b
(°C)
49.88 3.10 —3.73 25.40
53.49 3.60 —3.53 24.80
50.48 2.90 —3.37 28.40
49.42 3.30 —4.08 23.70
55.00 4.20 —3.80 29.80
52.65 3.20 —3.29 23.80
52.94 3.80 —3.84 27.80
51.82 3.80 —4.09 27.50
51.53 3.30 5.40 3.61 24.70
54.70 3.70 6.39 3.40 26.90
56.14 4.30 3.02 3.67 27.20
50.00 3.20 6.27 3.82 19.30
50.18 4.00 7.60 4.73 27.30
55.11 4.00 5.65 3.60 25.10
57.13 4.10 3.19 3.32 27.70
56.06 4.60 3.47 3.94 23.70
57.00 4.80 5.82 3.91 27.40
53.15 ± 2.66 3.76 ± 0.55 5.20 ± 1.61 3.75 ± 0.36 25.79 ± 2.38
Note: The em dashes correspond to control individuals and the values in the
last row are means ± SD.
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thermoregulatory lizard would attain in the environment or if
corrections were needed (sensu Kubisch et al. 2016).
To determine the thermal quality of the potential refuges, we
applied the concept of degree-days (sensu Lindsey and Newman
1956), using as reference the values 0 °C (the melting point of
water at 1 atm (1 atm = 101.325 kPa)) and 5 °C (the mean of the
CTmin for this species). Degree-days are the summation of tem-
perature differences to a reference value over time. In this way,
the degree-days explain both the magnitude and the duration of
temperatures that lizards would experience in relation to a single
parameter (the reference chosen value). This metric allows a di-
rect comparison of thermal regimes among different sites for
many species or species populations (Guisan and Hofer 2003;
Schwanz and Janzen 2008;Murphy et al. 2010;Boyero et al. 2011;
Graae et al. 2012;Mitchell et al. 2012). In this study, the reference
values chosen were 0 and 5 °C to infer how often and how long
L. p. argentinus individuals would be subjected to temperatures
below the melting point of water and above the CTmin of the
species, respectively, during the autumn, winter, and spring
months. We used degree-days to compare among the different
potential refuges, which was calculated using the formula:
HRDD0 ⫽兺
i⫽1
n
|(Ti⫺0)/48|
where HRDD0 is the heating refuge degree-day for 0 °C and
T
i
refers to the registered temperature values below 0 °C (every
30 min) and
RDD5 ⫽兺
i⫽1
n
|(Ti⫺5)/48|
where RDD5 is the refuge degree-day for 5 °C and T
i
refers to the
registered temperature values above 5 °C (every 30 min).
Fig. 1. Operative temperatures obtained with the lizard models from autumn to spring in the two possible overwintering habitats of
Liolaemus pictus argentinus at different elevations of a mountain in Esquel, Argentina (42°49=S, 71°15=W): (A) forest (1100 m above sea level (asl))
and (B) highlands (1400 m asl). Color version online.
Cecchetto et al. 775
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Laboratory experiments
We brought the lizards to the laboratory where we measured
snout–vent length (SVL) and body mass (Table 1) using a digital
caliper (±0.02 mm) and Ohaus balance Scot Pro (±0.01 g), respec-
tively.
We kept lizards individually in cotton bags at room tempera-
ture (21 °C) for a maximum of 48 h. Before the cooling experi-
ments, we placed lizards in a refrigerator at 15 °C for 30 min.
The cooled down group was placed individually into dry plastic
containers positioned in a freezer, whereas a control group was
placed into the same conditions at room temperature (20 °C).
Temperatures of the cooled down group were regulated by adding
ice and table salt (NaCl) to the ice contained in the freezer to lower
the temperatures, or by adding water to attenuate cooling rates
when they were too high. We connected the lizards to a TC-08
Data Acquisition Module Omegas (8-Channel USB Thermocouple,
±0.01 °C) by ultra-thin (1 mm) catheter thermocouples to register
the body temperature in both groups during the cooling experi-
ment. A PVC pipe lizard model was set in another plastic con-
tainer and exposed to the same temperature fluctuations as the
lizards from the cooled down group.
We performed the experiment in three stages, considering that
lizards are normally exposed to air temperature fluctuations with
smooth drops and extended periods hovering near 0 °C in this
season (as seen in Z. vivipara;Grenot et al. 2000;Costanzo et al.
2008). In the first stage (from 20 to 0 °C), we exposed individuals to
cold from the experimental starting temperature (15 °C) to 0 °C for
6 h, at a rate of −0.12 °C/min. In the second stage (overnight),
lizards stayed at approximately 0 °C for the whole night (16 h).
Finally, in the third stage (below 0 °C), we dropped the tempera-
ture at a rate of −0.024 °C/min from 3.5 to 4 h until at least −3 °C
was reached (Figs. 2A,2B). Lizards stayed at the minimum temper-
ature for 0.5–1.5 h.
When computing cooling rates for analysis, in addition to raw
temperature values, we used an adjusted body temperature (AT
b
)
to standardize the change in temperature, considering that initial
Fig. 2. Five Liolaemus pictus argentinus individuals, a control, and a lizard model showing changes in (A) raw temperature and (B) adjusted body
temperature (AT
b
) for the three stages of the cooling experiment. Color version online.
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temperature values slightly varied among individuals. This index
illustrates the change in temperature independently from initial
values. We used the following formula:
ATb⫽[(Tb⫺Tbi)/Tbi] × 100
where T
b
is the body temperature at a given time and T
bi
is the
body temperature at the beginning of the experiment.
At the beginning of the experiments, we monitored lizards to
determine the CTmin (Table 1), defined as the temperature at the
lower extreme of tolerance at which the animal cannot right itself
when placed onto its back (i.e., the loss of righting response sensu
Doughty 1994). We evaluated CTmin by quickly taking lizards out
of the containers and placing them on their back as soon as they
started reaching values of ⬃10 °C. If the animal was able to right
itself, then it was placed back to continue cooling and the process
was repeated until the CTmin value was found. To control for a
potential effect of the handling on the individuals, such as a re-
lease of glucose caused by a sympathetic response, we handled all
control individuals in the same way as the treatment group.
At the end of the experiments, lizards were sacrificed by decap-
itation immediately after their extraction from the containers.
Liver and heart samples of each individual were then collected
into Eppendorf tubes and refrigerated at 2 °C until they were
analyzed the next day.
Biochemical analysis
We combined liver and heart samples of each individual into a
single Eppendorf tube per lizard due to their small volumes. We
homogenized all samples manually with a mortar and pestle, di-
luted with physiological saline (9% v/v) in a 1:4 dilution, and then
placed them in Eppendorf tubes to be centrifuged at 3200 r/min
for 10 min. Afterwards, we used tubes in an absorption spectros-
copy test with enzymatic assays, testing for urea, total proteins,
and albumin. We made the selection of the cryoprotectants ana-
lyzed in this study considering biochemical variables found rele-
vant in previous studies of cold hardening in reptiles (for urea,
glucose, and antifreeze proteins (AFPs), respectively: Costanzo
et al. 2000;Grenot et al. 2000;Voituron et al. 2002) and in other
taxa (for AFPs: Storey and Storey 1986). We inferred the presence
of AFPs by considering the differences between total proteins and
albumin in the homogenate, taking into account that an increase
in total proteins without a corresponding increase in albumin
would point to proteins related to the cooling experiment (al-
though not necessarily AFPs). We determined all parameters for
the supernatant using a Clinical Chemistry Analyzer INCCA ver-
sion 2.05.01 (DICONEX) with an absorption spectroscopy test with
enzymatic assays and chemical reagents (Wiener Lab, Rosario,
Argentina). The kits used were kinetic urea UV AA, total proteins
AA, and albumin AA. We previously reprogrammed the biochem-
ical kit methods in relation to proportions and calibration values
to include sample values into the standard calibration curve and
to obtain reliable results.
Glucose
We measured glucose by taking a drop of blood from the caudal
vein near the cloaca, before and after the experiment, using a
glucometer (Accu-Chek®Performa Nano, with a range of 10 to
600 mg/dL), following the methodology of Voituron et al. (2002).
The small volumes of blood that we could obtain from lizards
without harming the animals only permitted one measurement
on each individual.
We calculated the glucose change (%) or adjusted glucose
change (AGluc) to account for the difference in glucose initial
values, given their uneven diet coming from the field, using the
following formula:
AGluc ⫽[(Gluc_f ⫺Gluc_i)/Gluc_i] × 100
where Gluc_f was the glucose at the end of the experiment and
Gluc_i was the glucose at the beginning.
Statistical analyses
We made comparisons of glucose for each individual before and
after the cooling experiment using a paired ttest and posterior
comparisons using ANCOVAs between control and experimental
groups, with the body condition index (BCI) (Peig and Green 2009)
as a covariable. We calculated the BCI using the package lmodel2
(Legendre 2014)inR(R Core Team 2017), and all other analyses
using the same software, with the nlme (Pinheiro et al. 2017) and
car (Fox and Weisberg 2011) packages.
BCI was calculated as
BCI ⫽Mi× (SVL0/SVLi)bSMA
where M
i
and SVL
i
are the mass and SVL of the individual, SVL
0
is
the arithmetic mean SVL of the population, and b
SMA
is the stan-
dardized major axis slope from the regression of ln body mass on
ln SVL for the population (Peig and Green 2009,2010). We calcu-
lated the b
SMA
exponent using the package lmodel2 (Legendre
2014)inR(R Core Team 2017).
The significance threshold for pvalues was set at 0.05.
Results
Body size (SVL), mass, CTmin, and BCI
Body size of L. p. argentinus ranged from 49.42 to 57.13 mm and
body mass ranged from 2.9 to 4.8 g. Lizards showed a CTmin
ranging from 3.02 to 7.6 °C. There were no significant differences
in the BCI between control individuals (3.72 ± 0.30, mean ± SD)
and cooled down individuals (3.78 ± 0.41, mean ± SD) (ANOVA:
F
[1,15]
= 0.12, p= 0.74; Table 1).
Annual air temperature, T
b
, thermal microenvironments,
and operative temperatures in the field (degree-days)
Air temperature at 1000 m asl reached minimum mean daily
values of −3.75 °C, whereas the lowest mean daily temperature
registered by the lizard PVC models was −1.7 °C. The lowest abso-
lute value registered by the lizard PVC models was −3 °C (Figs. 1A,1B).
T
b
was 25.79 ± 2.38 °C, whereas temperature in April (month of
capture) was 7.00 ± 1.59 °C at the low-elevation refuges and 10.41 ±
2.18 °C at the high-elevation refuges (Fig. 3).
The number of consecutive hours that each model recorded
temperatures below 0 °C varied between the different potential
refuges, from 0 to 28 times, and between 0.5 and 377.5 h below
0°C(Table 2).
In the heating refuge degree-day for 0 °C, the refuge with less
degree-days below 0 °C was buried ⬃10 cm under a tree, at 1100 m
asl (0 degree-days); and the refuge with the most degree-days be-
low 0 °C was buried ⬃10 cm under a dried log, at 1100 m asl
(18.25 degree-days) (Fig. 4). In the refuge degree-day for 5 °C, the
distribution of degree-days was homogeneous among potential
refuges at each site, with a larger amount at 1400 m asl (195.42 ±
6.07 degree-days) than at 1100 m asl (56.74 ± 8.02 degree-days)
(Table 2).
Control and cooled down individuals before and after
the experiments
During the cooling experiment, we did not detect an exother-
mic reaction from any individual and, after removing lizards from
the plastic containers, we found no ice or evident sign of freezing
(such as rigidity of the animals or a change in the color of their
skin). Additionally, individuals reacted seconds after we took
them out of the freezer, although in a seemingly lethargic state,
with slow movements.
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The control individuals showed negative values of adjusted glu-
cose change (mean = −14.6%) and individuals that were cooled
down showed positive values (mean = 18.9%), using BCI as a signif-
icant covariable (ANCOVA: F
[1,15]
= 6.43, p= 0.02; Fig. 3).
The urea, total proteins, and albumin, which were measured
only after the experiments, did not show significant differences
between the control and the cooled down individuals (Table 3).
Discussion
In the subantarctic forest of Nothofagus pumilio (Poepp. & Endl.)
Krasser (Cabrera 1971), L. p. argentinus can choose refuges to endure
the cold environmental conditions without resorting to big phys-
iological investments like freeze tolerance or supercooling. The
physiological cold hardiness mechanisms are energetically costly
because glucose, lipids, and molecules involved in the synthesis of
proteins used for these mechanisms cannot be allocated for other
metabolic processes. Liolaemus pictus argentinus did not show a
detectable exothermic reaction when exposed to below-zero tem-
perature values, suggesting that there was no freezing of a signif-
icant amount of tissue or plasma. Furthermore, all lizards
survived the experiments and started to move seconds after we
exposed them to temperatures equal or lower to the lowest value
found in the potential refuges, which might suggest a capacity for
supercooling.
The long period of brumation (winter dormancy in ectothermic
vertebrates, sensu Mayhew 1965) that reptiles in Patagonia expe-
rience reduces the period of activity, affecting multiple aspects of
their life history, such as growth (Gutiérrez et al. 2013) and repro-
ductive cycles (Ibargüengoytía and Cussac 1996). Given that the
allocation of energy is vital to compensate for the slow lives of
Patagonian lizards (Boretto et al. 2018), behavioral mechanisms
seem better suited for the survival of L. p. argentinus than physio-
logical mechanisms.
Between the mid-elevation forest and the limit with the high
Andean steppes, L. p. argentinus lizards can find refuges where they
would spend little to no time at subzero temperatures; further-
more, elevation proved to be a weak predictor of minimum tem-
perature in areas with varied potential refuges available (Fig. 4)
(Lookingbill and Urban 2003;Dobrowski et al. 2009). Unexpect-
edly, the temperatures in the potential refuges at the forest at
mid-elevations were sometimes lower than temperatures at the
high-elevation site, especially during spring and autumn (Figs. 1A,
1B), and both sites spent little to no time below 0 °C, even in
mid-winter (Fig. 4,Table 2). The forests cool down the soil surface
by means of direct shading and, indirectly, through evapotrans-
piration in the hotter days of spring and autumn. However, when
the snow starts to accumulate during winter, it keeps the soil
surface warmer than the air temperature. During the spring, the
Fig. 3. Temperatures of potential refuges in the mid-elevation forest (1100 m above sea level (asl)) and the high-elevation site (1400 m asl) for
the month of capture (April) and field body temperature (T
b
)ofLiolaemus pictus argentinus. Median (black horizontal lines) and mean (diamonds)
values are shown for all groups. The middle 50% of values are inside each box and the whiskers represent upper and lower quartiles; the
symbols beyond the whiskers are outliers.
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snow both reflects incoming radiation and absorbs latent heat
during the melting process, which keeps the soil colder (Graae
et al. 2012). Therefore, after the snow begins to melt, the canopy
would protect the refuges in the forest, whereas the refuges at the
high-elevation site would be more vulnerable. Moreover, the op-
erative temperatures of the potential refuges show that there are
notable differences in the episodes when temperature drops be-
low 0 °C, the duration of such episodes, and the range of the
available temperatures among the potential refuges. This differ-
ence could reflect a very different threat of freezing and thawing
episodes for the lizards, depending on the chosen refuge and the
elevation. The high variation of viable potential refuges at the
mid-elevation forest and the high-elevation site, and within each
site, implies that organisms could in theory escape freezing by
choosing a refuge, with the possibility of relocating by moving
short distances (i.e., for a few metres when temperatures are
above CTmin).
Even though choosing a high-elevation refuge might be more
dangerous than a mid-elevation refuge during mid-winter, in au-
tumn and spring, they offer the possibility of short bursts of ac-
tivity. We found that the amount of degree-days above 5 °C was
over three times greater at the high-elevation site (Fig. 4), mean-
ing that lizards choosing refuges at such elevations spend more
time above the mean CTmin for the species. This would enable
L. p. argentinus in those refuges to take better advantage of the
occasional sunny days available in the coldest months of the year
for activities such as foraging, escaping predators, or moving to
another refuge, as was the case for the Zodiac Tree Iguana (Liolaemus
signifer (Duméril and Bibron, 1837)) (Pearson 1954). Therefore, it is
feasible that even in Patagonia, lizards can survive winter mainly
by choosing appropriate refuges that allow them to avoid facing
temperatures below zero.
Although results are preliminary, we did not find any signifi-
cant differences between control and cooled down individuals for
total proteins or for albumin, even though AFPs occur in various
organisms relying on supercooling to survive exposure to sub-
freezing temperatures (Devries 1982). Nevertheless, although
AFPs seem to be quite important in the survival of some polar
fishes (DeVries 1988), they have not been found in the blood of
freeze-tolerant Wood Frogs (Lithobates sylvaticus (LeConte, 1825);
previously Rana sylvatica LeConte,1825) (Wolanczyk et al. 1990),
nor in hatchlings of the Eastern Painted Turtle (Chrysemys picta
(Schneider, 1783)) (Storey et al. 1991), nor in the Snapping Turtle
(Chelydra serpentina (Linnaeus, 1758)) (Costanzo et al. 2000). It
should be noted that there is scarce information regarding AFPs in
lizards, making it difficult to estimate the concentration of poten-
tial AFPs in L. p. argentinus. Considering results found in fish
(Wilson et al. 2010), where the concentration of type I AFPs was
10–35 mg/mL, the technique used in this study might not have
enough resolution to detect a potential increase in AFPs.
Consistent with the interest that motivated the analyses of total
proteins and albumin, we analyzed lizards subjected to freezing
temperatures for urea, which is the primary catabolic product of
protein metabolism. In addition, high concentrations of urea
were observed in a response related to freeze avoidance (by incre-
menting the plasma osmolality) in hatchlings of C. picta (an in-
crease of 60–70 mosmol/L, mainly attributed to urea) after
acclimation to simulated winter conditions (Costanzo et al. 2000).
However, no differences were found in the urea between cooled
down and control individuals of L. p. argentinus under experimen-
tal conditions.
The increase in blood glucose in cooled down lizards, in con-
trast with the decrease found in controls, might indicate that
L. p. argentinus is using this metabolite as a fast physiological an-
tifreeze response to cold temperatures. However, given that the
increase in glucose found in other species was proportionally
much larger (Grenot et al. 2000;Costanzo et al. 2008;Doucet et al.
2009), it is most likely that the role glucose plays in the cold
Table 2. Comparison between data obtained from six lizard models set in both possible locations of Liolaemus pictus argentinus for overwintering.
Forest site (1100 m above sea level (asl)) High Andean site (1400 m asl)
Model 1 under a
dead tree, buried
⬃10 cm
Model 2 under a
dried log, buried
⬃10 cm
Model 3 under a
tree, buried
⬃10 cm
Model 1 under a tussock
(Mulinum spinosum), near
the roots (⬃10 cm)
Model 2 under the ground
(⬃15 cm), beneath a shrub
(Berberis sp.)
Model 3 under the center
of a large boulder
(⬃2 m diameter)
Number of times exposed to temperatures below 0 °C 1 15 0 28 8 19
Mean (±SD) consecutive hours exposed to temperatures
below 0 °C (h)
146.50 60.25±97.00 —11.00±23.00 7.25±6.00 10.50±8.00
Minimum to maximum consecutive hours exposed to
temperatures below 0 °C (h)
146.50 1.50 to 377.50 —0.50 to 93.50 0.50 to 16.00 0.50 to 20.50
Heating refuge degree-days below 0 °C (degree-days) 1.53 18.25 0 11.21 1.40 5.36
Refuge degree-days above 5 °C (degree-days) 50.83 65.87 53.53 202.29 193.19 190.78
Minimum to maximum temperatures (°C) −0.68 to 10.15 −2.28 to 11.37 0.25 to 9.34 −2.19 to 22.66 −0.99 to 23.01 −1.73 to 21.51
Cecchetto et al. 779
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hardiness of this species, if any, is not vital to its survival or at least
not directly related to freeze-point depression.
The simplicity of the glucose synthesis pathway, plus the natu-
ral mechanisms for rapidly activating liver glycogenolysis in ver-
tebrates, are probably the primary reasons for the use of glucose
as a cryoprotectant (Storey 1990). The role of glucose as a cryopro-
tectant and as a solute colligatively lowering the freezing point
of body fluids has been considered in reptile species such as
Z. vivipara (Grenot et al. 2000) and C. picta (Costanzo et al. 1995b), as
well as in amphibian species such as L. sylvaticus and Spring Peeper
(Pseudacris crucifer (Wied-Neuwied, 1838)) (Storey and Storey 1986).
Glucose clearance may be necessary afterwards because of the
many negative effects of sustained high glucose on metabolism,
such as those that occur with diabetes (Storey and Storey 1996).
However, glucose can also play a role as a metabolic fuel during
anaerobic metabolism during extended freezing periods (Calderon
et al. 2009;Sinclair et al. 2013).
How lizards can endure situations in which there is snow or
rain most of the year with temperatures dropping rapidly during
the night even in summer is a fascinating subject to study, and the
mountains of Patagonia represent an ideal location to do so. Even
living in these challenging environments, L. p. argentinus did not
show any notable physiological mechanisms, only a small in-
crease in blood glucose that is not likely related to freeze-point
depression. Considering the very low environmental tempera-
tures that these lizards face most of the year, the ability to over-
come the punitive environmental constraints must rely mainly in
appropriate refuges. However, considering lizards survived the
temperatures of our experiments without freezing, L. p. argentinus
might be able to develop supercooling under extreme tempera-
tures, given that some potential refuges dropped below 0 °C mul-
tiple times. Appropriate refuges can give individuals an ecological
advantage in cold seasons when some intermittent hot days might
occur, thus enabling them to resume activity when other species
may still be undergoing torpor. This work provides insights into
the dynamic interplay between behavioral strategies and physio-
logical mechanisms that animals must balance to survive subzero
temperatures.
Fig. 4. Thermal quality of the potential refuges of Liolaemus pictus argentinus (degree-day) in the mid-elevation forest (1100 m above sea level
(asl)) and the high-elevation site (1400 m asl). Values for degree-days below 0 °C (light gray) and degree-days above 5 °C (dark gray) are shown
for each potential refuge.
Table 3. Comparison of biochemical variables (concentrations expressed in g/L; mean ± SD)
between control (n= 8) and cooled down (n= 9) individuals of Liolaemus pictus argentinus.
Individuals
Control Cooled down df Fp
Urea 3.468±1.558 3.253±2.027 1, 10 1.472 0.247
Initial glucose 1.376±0.114 1.437±0.260 —— —
Final glucose 1.158±0.222 1.644±0.205 —— —
Total proteins 184.625±45.904 215.000±123.414 1, 10 0.472 0.492
Albumin 38.150±7.153 47.156±14.399 1, 10 0.567 0.451
Note: Analyses were performed as ANCOVAS with body condition index (BCI) as a covariable. Initial
and final glucose concentrations were not analyzed because the change in glucose was analyzed as
AGluc (%). For initial glucose concentration, all individuals were taken into account in the comparison
(n= 25). In the case of the urea concentration, values include the significant covariable BCI (F
[1,10]
=
10.356, p= 0.001).
780 Can. J. Zool. Vol. 97, 2019
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Acknowledgements
We thank F. Duran for his help in the field, capturing lizards.
The group also thanks J.M. Carbajalino Fernández and C. Navas for
their insightful comments on the methodology and F. Baudino for
her support during the experiments. This study was conducted
with research grants from Fondo para la Investigación Científica y
Tecnológica (PICT-2014-3100) and Consejo Nacional de Investiga-
ciones Científicas y Técnicas (PIP-11220120100676).
References
Berman, D.I., Leirikh, A.N., and Mikhailova, E.I. 1984. Winter hibernation of the
Siberian salamander Hynobius keyserlingi. J. Evol. Biochem. Physiol. 3(1–2):
323–327.
Berman, D.I., Bulakhova, N.A., Alfimov, A.V., and Meshcheryakova, E.N. 2016.
How the most northern lizard, Zootoca vivipara, overwinters in Siberia. Polar
Biol. 39(12): 2411–2425. doi:10.1007/s00300-016-1916-z.
Boretto, J.M., Cabezas-Cartes, F., and Ibargüengoytía, N.R. 2018. Slow life histo-
ries in lizards living in the highlands of the Andes Mountains. J. Comp.
Physiol. B, 188(3): 491–503. doi:10.1007/s00360-017-1136-z. PMID:29150716.
Boyero, L., Pearson, R.G., Gessner, M.O., Barmuta, L.A., Ferreira, V., Graça, M.A.S.,
et al. 2011. A global experiment suggests climate warming will not accelerate
litter decomposition in streams but might reduce carbon sequestration. Ecol.
Lett. 14(3): 289–294. doi:10.1111/j.1461-0248.2010.01578.x. PMID:21299824.
Cabrera, A.L. 1971. Fitogeografía de la República Argentina. Bol. Soc. Argentina
Bot. 14: 1–42.
Calderon, S., Holmstrup, M., Westh, P., and Overgaard, J. 2009. Dual roles of
glucose in the freeze-tolerant earthworm Dendrobaena octaedra: cryoprotec-
tion and fuel for metabolism. J. Exp. Biol. 212(Pt. 6): 859– 866. doi:10.1242/jeb.
026864. PMID:19252003.
Christian, K.A., and Tracy, C.R. 1981. The effect of the thermal environment on
the ability of hatchling Galapagos land iguanas to avoid predation during
dispersal. Oecologia, 49(2): 218–223. doi:10.1007/BF00349191. PMID:28309312.
Claussen, D.L., Townsley, M.D., and Bausch, R.G. 1990. Supercooling and freeze-
tolerance in the European wall lizard, Podarcis muralis, with a revisional his-
tory of the discovery of freeze-tolerance in vertebrates. J. Comp. Physiol. B,
160(2): 137–143. doi:10.1007/BF00300945.
Colwell, R.K., Brehm, G., Cardelús, C.L., Gilman, A.C., and Longino, J.T. 2008.
Global warming, elevational range shifts, and lowland biotic attrition in the
wet tropics. Science, 322(5899): 258–261. doi:10.1126/science.1162547. PMID:
18845754.
Costanzo, J.P., Claussen, D.L., and Lee, R.J. 1988. Natural freeze tolerance in a
reptile. Cryo-Letters, 5(6): 5–10.
Costanzo, J.P., Iverson, J.B., Wright, M.F., and Lee, R.E. 1995a. Cold hardiness and
overwintering strategies of hatchlings in an assemblage of northern turtles.
Ecology, 76(6): 1772–1785. doi:10.2307/1940709.
Costanzo, J.P., Lee, R.E., Devries, A.L., Wang, T., and Layne, J.R. 1995b. Survival
mechanisms of vertebrate at subfreezing temperatures: applications in
cryomedicine. FASEB J. 9(5): 351–358. doi:10.1096/fasebj.9.5.7896003. PMID:
7896003.
Costanzo, J.P., Litzgus, J.D., Iverson, J.B., and Lee, R.E. 2000. Seasonal changes in
physiology and development of cold hardiness in the hatchling painted tur-
tle Chrysemys picta. J. Exp. Biol. 203(Pt. 22): 3459–3470. PMID:11044384.
Costanzo, J.P., Lee, R.E., and Ultsch, G.R. 2008. Physiological ecology of overwin-
tering in hatchling turtles. J. Exp. Zool. Part A Ecol. Genet. Physiol. 309(6):
297–379. doi:10.1002/jez.460.
Cowles, R.B., and Bogert, C.M. 1944. A preliminary study of the thermal require-
ments of desert reptiles. Bull. Am. Mus. Nat. Hist. 83(5): 265–296.
Devries, A.L. 1982. Biological antifreeze agents in coldwater fishes. Comp.
Biochem. Physiol. Part A, 73(4): 627–640. doi:10.1016/0300-9629(82)90270-5.
DeVries, A.L. 1988. The role of antifreeze glycopeptides and peptides in the
freezing avoidance of Antarctic fishes. Comp. Biochem. Physiol. Part B, 90(3):
611–621. doi:10.1016/0305-0491(88)90302-1.
Dobrowski, S.Z., Abatzoglou, J.T., Greenberg, J.A., and Schladow, S.G. 2009. How
much influence does landscape-scale physiography have on air temperature
in a mountain environment? Agric. For. Meteorol. 149(10): 1751–1758. doi:10.
1016/j.agrformet.2009.06.006.
Doucet, D., Walker, V.K., and Qin, W. 2009. The bugs that came in from the cold:
molecular adaptations to low temperatures in insects. Cell. Mol. Life Sci.
66(8): 1404–1418. doi:10.1007/s00018-009-8320-6. PMID:19129970.
Doughty, P. 1994. Critical thermal minima of garter snakes (Thamnophis) depend
on species and body size. Copeia, 1994(2): 537–540. doi:10.2307/1447008.
Espinoza, R.E., and Tracy, C.E. 1997. Thermal biology, metabolism, and hiberna-
tion. In Biology, husbandry, and health care of reptiles. 1st ed. Edited by
L. Ackerman. TFH Publications Inc., Neptune City, N.J. pp. 159–194.
Fox, J., and Weisberg, S. 2011. An R companion to applied regression. 2nd ed.
Sage, Thousand Oaks, Calif.
Graae, B.J., De Frenne, P., Kolb, A., Brunet, J., Chabrerie, O., Verheyen, K., et al.
2012. On the use of weather data in ecological studies along altitudinal and
latitudinal gradients. Oikos, 121(1): 3–19. doi:10.1111/j.1600-0706.2011.19694.x.
Grenot, C.J., Garcin, L., Dao, J., Hérold, J.P., Fahys, B., and Tséré-Pagès, H. 2000.
How does the European common lizard, Lacerta vivipara, survive the cold of
winter? Comp. Biochem. Physiol. A, 127(1): 71–80. doi:10.1016/S1095-6433(00)
00236-1.
Guisan, A., and Hofer, U. 2003. Predicting reptile distributions at the mesoscale:
relation to climate and topography. J. Biogeogr. 30(8): 1233–1243. doi:10.1046/
j.1365-2699.2003.00914.x.
Gutiérrez, J.A., Piantoni, C., and Ibargüengoytía, N.R. 2013. Altitudinal effects on
life history parameters in populations of Liolaemus pictus argentinus (Sauria:
Liolaemidae). Acta Herpetol. 8(1): 9–17. doi:10.13128/Acta_Herpetol-11056.
Ibargüengoytía, N.R. 2005. Field, selected body temperature and thermal toler-
ance of the syntopic lizards Phymaturus patagonicus and Liolaemus elongatus
(Iguania: Liolaemidae). J. Arid Environ. 62: 435–448. doi:10.1016/j.jaridenv.
2005.01.008.
Ibargüengoytía, N.R., and Cussac, V.E. 1996. Reproductive biology of the vivipa-
rous lizard, Liolaemus pictus (Tropiduridae): biennial female reproductive cir-
cle? Herpetol. J. 6: 137–143.
Körner, C. 2007. The use of ‘altitude’ in ecological research. Trends Ecol. Evol.
22(11): 569–574. doi:10.1016/j.tree.2007.09.006. PMID:17988759.
Kubisch, E.L., Corbalán, V., Ibargüengoytía, N.R., and Sinervo, B. 2016. Local
extinction risk of three species of lizard from Patagonia as a result of global
warming. Can. J. Zool. 94(1): 49–59. doi:10.1139/cjz-2015-0024.
Legendre, P. 2014. lmodel2: Model II regression. Available from https://CRAN.R-
project.org/package=lmodel2.
Lindsey, A.A., and Newman, J.E. 1956. Use of official weather data in spring time:
temperature analysis of an Indiana phenological record. Ecology, 37(4): 812–
823. doi:10.2307/1933072.
Lookingbill, T.R., and Urban, D.L. 2003. Spatial estimation of air temperature
differences for landscape-scale studies in montane environments. Agric. For.
Meteorol. 114(3–4): 141–151. doi:10.1016/S0168-1923(02)00196-X.
Mayhew, W.W. 1965. Hibernation in the horned lizard, Phrynosoma m’calli. Comp.
Biochem. Physiol. 16(1): 103–119. doi:10.1016/0010-406X(65)90167-2. PMID:
5861527.
Mitchell, N., Hipsey, M., Arnall, S., McGrath, G., Tareque, H., Kuchling, G.,
Vogwill, R., Sivapalan, M., Porter, W., and Kearney, M. 2012. Linking eco-
energetics and eco-hydrology to select sites for the assisted colonization of
Australia’s rarest reptile. Biology (Basel), 2(1): 1–25. doi:10.3390/biology2010001.
PMID:24832649.
Monasterio, C., Salvador, A., Iraeta, P., and Díaz, J.A. 2009. The effects of thermal
biology and refuge availability on the restricted distribution of an alpine
lizard. J. Biogeogr. 36(9): 1673–1684. doi:10.1111/j.1365-2699.2009.02113.x.
Murphy, M.A., Evans, J.S., and Storfer, A. 2010. Quantifying Bufo boreas connec-
tivity in Yellowstone National Park with landscape genetics. Ecology, 91(1):
252–261. doi:10.1890/08-0879.1. PMID:20380214.
Pearson, O.P. 1954. Habits of the lizard Liolaemus m. multiformus at high altitudes
in southern Peru. Copeia, 1954(2): 111–116.
Peig, J., and Green, A.J. 2009. New perspectives for estimating body condition
from mass/length data: the scaled mass index as an alternative method.
Oikos, 118(12): 1883–1891. doi:10.1111/j.1600-0706.2009.17643.x.
Peig, J., and Green, A.J. 2010. The paradigm of body condition: a critical reap-
praisal of current methods based on mass and length. Func. Ecol. 24(6):
1323–1332. doi:10.1111/j.1365-2435.2010.01751.x.
Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., and R Core Team. 2017. nlme: Linear
and nonlinear mixed effects models. Available from https://CRAN.R-
project.org/package=nlme.
R Core Team. 2017. R: a language and environment for statistical computing.
R Foundation for Statistical Computing, Vienna, Austria. Available from
https://www.r-project.org/.
Salt, R.W. 1966. Factors influencing nucleation in supercooled insects. Can. J.
Zool. 44(1): 117–133. doi:10.1139/z66-009.
Schmid, W.D. 1982. Survival of frogs in low temperature. Science, 215(4533):
697–698. doi:10.1126/science.7058335. PMID:7058335.
Schwanz, L.E., and Janzen, F.J. 2008. Climate change and temperature-
dependent sex determination: can individual plasticity in nesting phenol-
ogy prevent extreme sex ratios? Physiol. Biochem. Zool. 81(6): 826–834.
doi:10.1086/590220. PMID:18831689.
Sinclair, B.J., Stinziano, J.R., Williams, C.M., Macmillan, H.A., Marshall, K.E., and
Storey, K.B. 2013. Real-time measurement of metabolic rate during freezing
and thawing of the wood frog, Rana sylvatica: implications for overwinter
energy use. J. Exp. Biol. 216: 292–302. doi:10.1242/jeb.076331. PMID:23255194.
Sømme, L. 1982. Supercooling and winter survival in terrestrial arthropods.
Comp. Biochem. Physiol. A, 73(4): 519– 543. doi:10.1016/0300-9629(82)90260-2.
Storey, B. 1990. Life in a frozen state: adaptive strategies for natural freeze
tolerance in amphibians and reptiles. Am. J. Physiol. 258(3 Pt. 2): R559–R568.
PMID:2180324.
Storey, K.B., and Storey, J.M. 1986. Freeze tolerance and intolerance as strategies
of winter survival in terrestrially-hibernating amphibians. Comp. Biochem.
Physiol. 83(4): 613–617. doi:10.1016/0300-9629(86)90699-7.
Storey, K.B., and Storey, J.M. 1996. Natural freezing survival in animals. Annu.
Rev. Ecol. Syst. 27(1): 365–386. doi:10.1146/annurev.ecolsys.27.1.365.
Storey, K.B., McDonald, D.G., Duman, J.G., and Storey, J.M. 1991. Blood chemistry
and ice nucleating activity in hatchling painted turtles. Cryo-Letters, 12(6):
351–358.
Voituron, Y., Storey, J.M., Grenot, C., and Storey, K.B. 2002. Freezing survival,
body ice content and blood composition of the freeze-tolerant European
Cecchetto et al. 781
Published by NRC Research Press
Can. J. Zool. Downloaded from www.nrcresearchpress.com by Dr. Marlin Medina on 10/07/19
For personal use only.

common lizard, Lacerta vivipara. J. Comp. Physiol. B, 172(1): 71–76. doi:10.1007/
s003600100228. PMID:11824405.
Weisrock, D.W., and Janzen, F.J. 1999. Thermal and fitness-related consequences
of nest location in Painted Turtles (Chrysemys picta). Funct. Ecol. 13(1): 94–101.
doi:10.1046/j.1365-2435.1999.00288.x.
Wilson, P.W., Osterday, K.E., Heneghan, A.F., and Haymet, A.D.J. 2010. Type I
antifreeze proteins enhance ice nucleation above certain concentrations.
J. Biol. Chem. 285(45): 34741–34745. doi:10.1074/jbc.M110.171983. PMID:
20837472.
Wolanczyk, J.P., Storey, K.B., and Baust, J.G. 1990. Ice nucleating activity in the
blood of the freeze-tolerant frog, Rana sylvatica. Cryobiology, 27: 328–335.
doi:10.1016/0011-2240(90)90032-Y. PMID:2379418.
782 Can. J. Zool. Vol. 97, 2019
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